Filoviruses are enveloped, nonsegmented, negative-stranded (NNS) RNA viruses and constitute a distinct family within the order Mononegavirales. The family includes Marburg viruses, causing Marburg disease (green monkey disease), and Ebola virus (EBOV), causing hemorrhagic fever. The Ebola viruses are further subdivided into four distinct African (Ivory Coast, Sudan, Zaire, and Bundibugyo) and a single Asian (Reston) species. EBOV Zaire (EboZ) and Sudan (EboS) are highly pathogenic in human and nonhuman primates, with a mortality rate up to 80-90%. Peters, C. J., et al., Curr. Top. Microbiol. Immunol., 235:85-95 (1999); Sanchez, A., et al. Filoviridae: Marburg and Ebola viruses, p. 1279-1304, D. M. Knipe and P. M. Howley (ed.), Fields virology, Lippincott, Williams & Wilkins, Philadelphia, Pa. (2001). In contrast, EBOV Ivory Coast (EboC), EBOV Bundibugyo, and EBOV Reston (EboR) are less virulent, with EboR infecting only non-human primates (Sanchez, A., et al., (2001) op. cit.). EBOV infections are pantropic, but no single organ shows sufficient damage to account for the onset of severe shock and bleeding. As with other viral hemorrhagic fevers, EBOV infections are associated with fluid distribution problems, hypotension, coagulation disorders and bleeding, finally resulting in fulminate shock.
Ebola virus is classified as a biosafety level-4 (BSL-4) agent because of its high mortality rate and the lack of approved vaccines and antivirals to prevent or treat it (Peters, C. J., et al., op. cit.; Sanchez, A., et al. (2001), op. cit.). EBOV has also been classified as a Category A bioweapons agent by the Centers for Disease Control and Prevention (CDC) because of its high virulence, demonstrated aerosol infectivity in the laboratory, and capacity for inducing fear and anxiety. See, Bossi, P., et al., Cell Mol. Life. Sci., 63:2196-212 (2006).
Several promising vaccine candidates have been shown to be effective in eliciting host immune responses and protecting primates against EBOV infection. Sullivan, N., et al., Nature, 424:681-684 (2000); Sullivan N., et al., J. Virol., 77:9733-9737 (2003). Nonetheless, the minimal time required for protective vaccination (more than one month), the sporadic nature of filoviral outbreaks and the potential for bioterrorism, underscore the urgent need to develop potent inhibitors for EBOV infection. Bray, M., Antiviral Res., 57:53-60 (2003).
There are currently no approved therapeutic interventions for EBOV infections. A limited number of small-molecule research inhibitors of EBOV infections have been reported to date. Bray M., et al., Antiviral Res., 54:1-17 (2002); Hensley, L. E., et al., Curr. Mol. Med., 5:761-72 (2005); Stroher, U., et al., Expert Opin. Investig. Drugs., 15:1523-35 (2006). These low molecular weight anti-EBOV agents can be characterized by three general modes of action: a) impairment of viral mRNA methylation; b) stimulation of innate antiviral mechanisms; and c) prevention of virion entry and/or fusion.
The carbocyclic adenosine analog 3-deazaadenosinc (C-c3Ado) has been shown to inhibit cellular S-adenosylhomocysteine hydrolase and EboZ replication in vitro, with an IC50 of 30 μM. Huggins, J., et al., J. Infect. Dis., 179:S240-7 (1999). The activity of C-c3Ado has been attributed to diminished methylation of the 5′ cap of viral mRNA by methyltransferase, which impairs the translation of viral transcripts. Administration of C-c3Ado to EBOV-infected mice has also been found to dramatically increase production of IFN-α and protects mice against EBOV infection. Huggins, J., et al., op. cit. However, C-c3Ado failed to induce IFN-α production and did not protect against EBOV infections in monkeys. Bray, M., et al., Antiviral Res., 55:151-9 (2002).
The glycodendritic compound, BH30sucMan, which contains 32 individual α-mannose units linked to the hyper-branched dendrimer BH30 through succinyl spacers, has recently been shown to block C-type lectins dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) mediated EBOV infection of Jurkat cells at high nanomolar concentrations (IC50=337 nM) (Lasala, F., et al., Antimicrob. Agents Chemother., 47:3970-2 (2003)).
EBOV enters target cells by an endocytic pathway. Therefore, compounds that disrupt the efficient internalization of the endosomal vesicles, via the various components of the cytoskeleton, or endosome acidification, can potentially abrogate EBOV entry and fusion into cells. Latrunculin and colchicine impair the formation of microfilaments and microtubules, respectively, and have been shown to inhibit the infection of EBOV GP pseudotypes (Yonezawa, A., et al., J. Virol., 79:918-26 (2005)). Similarly, HeLa cells pretreated with the bafilomycin A1 (Lasala, F., et al., op. cit.), an inhibitor of vacuolar ATPase, are shown to be resistant to infection by pseudotyped HIV type 1 virions (Yonezawa, A., et al., op. cit.).
Recently, two groups have independently demonstrated that cathepsin B (CatB) and cathepsin L (CatL) mediate viral entry by carrying out proteolysis of the EBOV GP 1 subunit. Chandran, K., et al., Science, 308:1643-1645 (2005); Schornberg, K., et al., J. Virol., 80:4174-8 (2006). Selective inhibitors of the CatB such as CA-074 or CA-074Me, were shown to greatly reduce the infectivity of EBOV pseudotypes. Unfortunately, given the demonstrated hypersensitivity of EBOV GP1 to digestion by other proteases, such as thermolysin, the clinical prospects for antiviral agents that solely target CatB and CatL is not encouraging. Jane-Valbuena, J., et al., J. Virol., 76:5184-97 (2002).
Interfering with the viral entry process is an attractive strategy for controlling viral infection. Entry of EBOV and other filoviruses into a host cell is mediated by a single viral glycoprotein (GP), a class I fusion protein. EBOV-GP consists of GP1 and GP2 subunits, which are linked by disulfide bonds and non-covalent interactions. GP1 is responsible for receptor binding and host tropism, while GP2 mediates viral/cell membrane fusion during viral entry.
Viral entry inhibitors can disrupt the viral life cycle and therefore prevent or treat infection. For example, enfuvirtide (marketed under the trade name Fuzeon® (also known as T-20) by Hoffmann-La Roche Ltd.) is a synthetic 36-amino-acid peptide that binds to a region of the envelope glycoprotein 41 of HIV type 1 (HIV-1) that is involved in the fusion of the virus with the membrane of CD4+ host cells. See, Wild, C., et al., AIDS Res. Hum. Retroviruses, 9:1051-3 (1993). However, T-20 has also highlighted the potential problems of peptidic antivirals that include lack of absorption from the gastrointestinal tract necessitating intravenous delivery and a high manufacturing cost.
Clearly, needs remain for new, potent inhibitors against EBOV and other filovirus infections. Inhibitors that could be used during natural outbreaks or bio-terrorist attacks, and that could be used either prophylactically to treat a potentially exposed population or therapeutically after exposure or infection, would be especially desirable.